Method for labeling the 3&#39; terminus of a synthetic oligonucleotide using a unique multifunctional controlled pore glass (MF-CPG) reagent in solid phase oligonucleotide synthesis

ABSTRACT

A method for derivatizing and labeling the 3&#39;-terminus of an oligonucleotide during solid phase synthesis comprising the use of a multifunctional reagent whose preferred structure is shown below. ##STR1## wherein CPG is controlled pore glass beads, Fmoc is 9-fluorenylmethyoxycarbonyl, and the alkylamine contains 1 to 50 carbon atoms.

This is a division of application Ser. No. 07/399,658, filed Aug. 28,1989, now U.S. Pat. No. 5,141,813.

BACKGROUND OF THE INVENTION

Single base substitutions in human genes are the cause or are stronglyassociated with a variety of human diseases such as thehemoglobinopathies (Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis,G. T. Horn, H. A. Erlich, and N. Arnheim [1985] Science 230: 1350-1354;Embury, S. H., S. J. Scharf, R. K. Saiki, M. A. Gholson, M. Colbus, N.Arnheim, and H. A. Erlich [1987] N. Engl. J. Med. 316: 656-660) andcancer (Liu, E., B. Hjelle, R. Morgan, F. Hecht, and J. M. Bishop [1987]Nature 330: 186-188; Rodenhuis, S., M. L. van de Werering, W. J. Mooi,S. G. Evers, N. van Zandwijk, and J. L. Bos [1987] N. Engl. J. Med. 317:929-935). Previously, if no convenient restriction sites were altered bythe base change, then the only recourse has been to clone and sequencethe affected gene. Recently, polymerase chain reaction (PCR)amplification of the DNA segment in question, coupled with hybridizationof specific oligonucleotide probes, has allowed sequence determinationwithout the need for molecular cloning. The applicability of the lattertechnique is dependent on the availability of versatile and inexpensiveoligonucleotide probes.

Methods to covalently attach labels and reporter molecules tooligonucleotides have permitted their use as non-radioactivehybridization probes. New technologies in non-isotopic gene probes(Agrawal, S., C. Christodoulou, and M. J. Gait [1986] Nucl. Acids Res.14: 6227-6245; Connolly, B. A. [1987] Nucl. Acids Res. 15: 3131-3139;Jablonski, E., E. W. Moomaw, R. H. Tullis, and J. L. Ruth [1986] Nucl.Acids Res. 14: 6115-6128; Haralambidis, J., M. Chai, and G. W. Tregear[1987] Nucl. Acids Res. 15: 4857-4876; Li, P., P. P. Medon, D. C.Skingle, J. A. Lanser, and R. H. Symons [1987] Nucl. Acids Res. 15:5275-5287), DNA sequencing analysis (Smith, L. M., S. Fung, M. W.Hunkapiller, T. J. Hunkapiller, and L. E. Hood [1985] Nucl. Acids Res.13: 2399-2412; Sproat, B. S., B. Beijer, P. Rider, and P. Neuner [1987]Nucl. Acids Res. 15: 4837-4848; Ansorge, W., B. Sproat, J. Stegemann, C.Schwager, and M. Zenke [1987] Nucl. Acids Res. 15: 4593-4602), electronmicroscopy (Sproat, B. S., B. Beijer, and P. Rider [1987] Nucl. AcidsRes. 15: 6181-6196), and X-ray crystallography (Sproat et al. [1987]Nucl. Acids Res. 15: 4837- 4848) have provided impetus for thedevelopment and improvement of such methods. As applications continue toemerge, more convenient oligonucleotide labeling techniques and reagentswill be required.

Current methods to introduce chemical modifications intooligonucleotides employ special phosphoramidite reagents during solidphase synthesis. Attention has focused on the 5' terminus and a numberof protected amino-alkyl phosphoramidites have been reported (Agrawal etal., supra; Connolly, supra; Jablonski et al., supra; Smith et al.,supra; Sproat et al. [1987] Nucl. Acids Res. 15: 6181-6196; Sinha, N. D.and R. M. Cook [1988] Nucl. Acids Res. 16: 2659-2669) to incorporate a5' terminal aliphatic primary amine. Oligonucleotides modified by thesereagents can be subsequently derivatized with fluorophores, biotin, andother molecules. Similarly, phosphoramidite reagents have also beendescribed which incorporate a thiol functionality on the 5' terminus(Sproat et al. [1987] Nucl. Acids Res. 15: 4837-4848; Ansorge et al.,supra; Connolly, B. A. [1985] Nucl. Acids Res. 13: 4484-4502).

Techniques modifying the 3' terminus are inconvenient and tedious.Lemaitre et al. (Lemaitre, M., B. Bayard, and B. Lebleu [1987] Proc.Natl. Acad. Sci. USA 84: 648-652; Lemaitre, M., C. Bisbal, B. Bayard,and B. Lebleu [1987] Nucleosides and Nucleotides 6: 311-315) havedescribed the attachment of a ribonucleoside to the 3' terminus of anoligonucleotide using T4 RNA ligase. Terminal 3' modification wasachieved after periodate-oxidation of the ribose ring followed byreductive amination. Another procedure by Zuckerman et al. (Zuckerman,R., D. Corey, and P. Schultz [1987] Nucl. Acids Res. 15: 5305-5321)incorporates a 3' terminal thiol group via solid phase oligonucleotidesynthesis. Although this procedure is more efficient, it requires manysynthetic steps and purifications. Thus, there remains a need for asimple and efficient method to synthesize 3' labeled oligonucleotides.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a reagent for use in solid phaseoligonucleotide synthesis having the following structure: ##STR2##wherein: C=carbon atom;

W=any solid such as controlled pore glass (CPG [CPG Biosupport, Inc.,Fairfield, N.J.]), alkylamine CPG, wherein alkyl can be from 1 to 50carbon atoms, and isomeric forms thereof, any chemical modification ofCPG, wherein the modification can be amines, hydroxyls, carboxyls,sulfhydryls, or disulfides, copolymers of styrene and divinylbenzene andany solid support stable to all the conditions of solid phaseoligonucleotide synthesis; W can also be a non-solid phosphoramiditegroup, --OP(OR₃)NR₄ R₅, wherein R₃ =--CH₃, --CH₂ CH₂ CN, or alkane of 1to 50 carbon atoms, inclusive, and isomeric forms thereof, and R₄, R₅=methyl, ethyl, isopropyl, or alkane as defined above (if W is anon-solid phosphoramidite group it is not restricted to the 3'terminus);

X=a cleavable linking arm connecting carbon C to W which can be anycombination of atom groups (e.g., --(CH₂)_(n) --, --OCO--, --CO₂,--NHCO--, --CONH--) that covalently connects to the solid phase (W)through a cleavable linkage, and is stable to all the conditions ofsolid phase oligonucleotide synthesis. Cleavable linkages include esterswhich are cleaved with ammonium hydroxide and disulfides which arecleaved with reducing agents. After oligonucleotide synthesis, cleavageof X from the solid phase results in the transfer of the entiremultifunctional linking arm to the 3' terminus of the synthesizedoligonucleotide. Preferably, X=--O--CO--CH₂ --CH₂ --CO-- (succinic acid)which is readily cleaved from the solid phase with ammonium hydroxide;

Y=a linking arm connecting carbon C to R₁ O-- that is at least onecarbon atom long and can be any combination of atom groups (e.g.,--(CH₂)_(n) --, --OCO--, --CO₂, --NHCO--, --CONH--) that covalentlyconnects to OR₁ and is stable to all the conditions of solid phaseoligonucleotide synthesis;

Z=a linking arm connecting carbon C to R₂ --A-- that is at least onecarbon atom long and can be any combination of atom groups (e.g.,--(CH₂)_(n) --, --OCO--, --CO₂, --NHCO--, --CONH--) that covalentlyconnects to AR₂ and is stable to all the conditions of solid phaseoligonucleotide synthesis;

R₁ O=a protected hydroxide group where R₁ is a base stable-acid labilehydroxyl protecting group, e.g., R₁ can be monomethoxytrityl (MMT) ordimethoxytrityl (DMT);

A=--NH--, --S--, or any functional group that is capable of attaching areporter molecule or any detectable complex;

R₂ =corresponding protecting group for A that is stable to all theconditions of solid phase oligonucleotide synthesis; and

B=H, --CH₃, --Z--A--R₂, --Y--OR₁, or any combination of atom groups thatare inert to solid phase oligonucleotide synthesis.

Specifically exemplified herein is MF-CPG®(3'Amine-ON CPG) wherein, withreference to (I) above, W=long chain alkyl amine CPG; X=--O--CO--CH₂--CH₂ --CO--; B=H; Y=--CH₂ --; Z=--CH₂ --; A=--NH--; R₂=9-fluorenylmethyl (Fmoc), trifluoroacetyl (TFA), or any baselabile-acid stable amine protecting group; and R₁ =DMT.

The structure of MF-CPG®(3' Amine-ON CPG) is as follows: ##STR3##wherein the alkyl of alkylamine can be from 1 to 50 carbon atoms,inclusive, and isomeric forms thereof.

The subject invention is useful in solid phase oligonucleotide synthesis(both oligodeoxyribonucleotide and oligonucleotide) to chemically modifythe 3' terminus of a synthetic oligonucleotide with any chemicalfunctional group. Useful functional groups are primary amines,sulfhydryls, disulfides, and any other group typically used forconjugation of reporter molecules. Also, the subject invention can beused for attaching a label to a functional group introduced at the 3'terminus of a synthetic oligonucleotide. Labels include any reportermolecules such as biotin, haptens, fluorophores, proteins, enzymes, andantibodies. Such modified and labeled oligonucleotide probes can be usedin any application where the said probe hybridizes to complementarysequences of a target polynucleotide. Further, the invention can be usedfor detecting low copy genes by using the polymerase chain reaction(PCR) to amplify the target gene segment and then employing theinvention for detecting the presence of specific polynucleotide insamples containing the same, biological samples, and, for example,clinical samples such as serum and blood. Still further, the subjectinvention can be used for the diagnosis of infectious diseases andpathogens, detection of oncogenes, diagnosis of genetic disorders, anddetection of point mutations or single base substitutions. The subjectinvention has utility in the areas of anti-sense molecular biology,electron microscopy, X-ray crystallography, and site-specific cleavageof DNA.

Thus, the subject invention concerns a novel multifunctional controlledpore glass reagent (MF-CPG®) (FIG. 1), useful to incorporate 3' terminalprimary aliphatic amines into synthetic oligonucleotides. MF-CPG®comprises a unique succinic acid linking arm which possesses both amasked primary amine for label attachment and a dimethoxytritylprotected hydroxyl for nucleotide chain elongation. Using MF-CPG®, asimple and convenient technique has been devised to attachnon-radioactive labels to the 3' terminus of oligonucleotides.Bifunctional probes can then be constructed by ³² P labeling the 5'terminus with T4 kinase and gamma ³² P-ATP. Using such bifunctionaloligonucleotide probes in conjunction with PCR amplification, a personcan detect a single base substitution in a target DNA segment.Exemplified herein is the detection of a single base substitution of thehuman H-ras protooncogene employing either functionality. The inventiontechnique thus expands the potential applications for oligonucleotidesas hybridization probes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The synthetic scheme for MF-CPG®.

FIGS. 2A and 2B: Analytical chromatograms of purified 3' biotinylatedH-ras oligonucleotide probes.

FIGS. 3(A-D): Detection of point mutations in the human H-ras gene usingspecific bifunctional oligonucleotide probes.

DETAILED DESCRIPTION OF THE INVENTION

Materials and Methods

Long chain alkylamine CPG was purchased from Pierce Chemical Co.Biotin-XX-NHS ester, PCR amplimers, and the Gene-tect Non-IsotopicDetection System were obtained from Clontech Laboratories, Inc. Taqpolymerase was acquired from Perkin Elmer Cetus. HPLC was performed on aRainin Rabbit HPX System using aquapore C8 reverse phase columns(Applied Biosystems, Inc.) for both preparative (100×10 mm) andanalytical (30×4.6 mm) runs. A Biosearch Cyclone DNA synthesizer wasused for oligonucleotide synthesis.

The subject invention comprises a novel and simple method to synthesize3' labeled oligonucleotides. In conventional solid phase DNA synthesis,the 3' terminal nucleotide is pre-attached to the CPG support from the3' hydroxyl through a succinimic acid linking arm, and theoligonucleotide is synthesized from the 5' hydroxyl by repetitive cyclesof chemical reactions. In the subject invention, a 3' primary aliphaticamine replaces the pre-attached nucleoside with a unique multifunctionalinking arm to give a multifunctional CPG, MF-CPG® (FIG. 1) whichtransfers a primary amine to the 3' terminus of a synthesizedoligonucleotide without changing any chemistry or adding extra steps.MF-CPG® possesses a uniquely engineered linking arm that complies withfour important criteria. First, the linking arm is attached to the CPGthrough an ester functionality that is readily cleaved with ammoniumhydroxide treatment. Second, the linking arm contains a masked primaryaliphatic amine which is acid stable and resistant to all the reagentsused in normal oligonucleotide synthesis. Third, in addition to beingacid stable, the amine protecting group is readily removed with ammoniumhydroxide treatment. Fourth, the linking arm contains a dimethoxytritylprotected primary hydroxyl group for oligonucleotide chain elongation.Hence, the method is fully adaptable to commercial DNA synthesizers andis as easy as synthesizing normal oligonucleotides.

Since a reporter molecule now can be easily attached to the 3' terminusof any oigonucleotide, both the 5' and 3' termini can be used to labelthe oligonucleotide. Such bifunctional oligonucleotide probes are bothsensitive and specific in detecting single base substitutions in targetDNA when used in conjunction with PCR. The sensitivity and specificityare the same regardless of the detection system; autoradiography orcolorimetric detection with a streptavidin-alkaline phosphataseconjugate. The convenience of using MF-CPG® to non-isotopically label anoligonucleotide at the 3' terminus with subsequent ³² P labeling at the5' terminus makes this reagent an attractive alternative to currentmethods of functionalizing oligonucleotides. Thus, the inventiontechnique expands the potential for applications employingfunctionalized oigonucleotides.

The preparation of MF-CPG® is outlined in FIG. 1.N-Fmoc-O-DMT-3-amino-1,2-propanediol was first derivatized with succinicanhydride. The carboxyl group of the succinylated derivative wasconverted to a p-nitrophenyl ester and directly reacted with long chainalkylamine CPG to give MF-CPG®. After capping, the loading capacity ofMF-CPG® was determined to be 27.8 μmol/g.

Two bifunctional oligonucleotide probes were constructed using MF-CPG®as follows: First, two 3' amino-modified oligonucleotides,GGCGCCGGCGGTGTGGGCAA-X (H-ras, wild type) and GGCGCCGGCGATGTGGGCAA-X(H-ras, codon 13 Asp) [X=3' primary amine modification], weresynthesized using MP-CPG. The coupling efficiency of the firstnucleotide, which is indicative of 3' primary amine incorporation,was >97% in both cases. After standard cleavage and deprotection withammonium hydroxide, the crude 3' amino-modified oligonucleotides werebiotinylated with Biotin-XX-NHS ester. The long linking arm ofBiotin-XX-NHS ester, a 14 atom spacer (XX) consisting of twoe-aminocaproic acid moieties linked in series, was used to make theblotins more available for detection. After size exclusion on SephadexG-25 (Pharmacia), the biotinylated oligonucleotides were purified bypreparative HPLC on a C8 reverse phase column. Analytical HPLCchromatograms are shown in FIG. 2. The presence of biotin was confirmedby a p-methylamino-cinnamaldehyde colorimetric test. Finally, theconstruction of the bifunctional probes was completed by ³² P labelingof the 5' ends with T4 kinase and gamma ³² P-ATP.

The applicability of these bifunctional oligonucleotide probes wasassessed in detecting single base substitutions in the H-rasprotooncogene. The probes used were specific either for the wild typeH-ras sequence at codon 13 (GGT) or for the transforming mutant sequenceat codon 13 (GAT) which substitutes aspartate for glycine. A 110-basepair segment which includes codon 13 of the H-ras gene was amplifiedfrom genomic DNA using the polymerase (PCR). By autoradiography (FIG.3B), the codon 13 aspartate probe hybridized only with the amplified DNAcarrying the H-ras codon 13 aspartate mutation and not with DNA fromnormal placenta, a transformed mouse fibroblast cell line (NIH3T3)harboring a mutant human H-ras gene with a codon 12 valine substitution(GTT), or with placental DNA amplified in the region surround N-rascodons 12 and 13. Hybridization with the wild type codon 13 H-ras probe(FIG. 3A), however, showed a signal only with amplified DNA from normalplacenta and the NIH3T3 cell line whose murine H-ras gene can also beamplified using our current amplimers. Cross hybridization between thehuman and mouse wild type H-ras genes was unexpected but may indicatethat the sequences probed are identical in the two species.

When the same hybridized filters were incubated withstreptavidin-alkaline phosphatase and5-bromo-4-chloro-3-indolyl-phosphate (BCIP), colorimetric signals wereseen over the same slots exhibiting a radiographic signal (FIG. 3C and3D). Thus, when used in conjunction with PCR, these bifunctional probesare both sensitive and specific in detecting single base pair mismatchesin target DNA. Furthermore, an oligonucleotide probe from a singleMF-CPG® preparation can be either biotinylated or radiolabeled withoutcompromising hybridization sensitivity.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

Example 1--Preparation of MF-CPG®

To a solution of N-Fmoc-O-DMT-3-amino-1,2-propanediol (2.2 g, 3.5 mmol)and 4-dimethylaminopyridine (200 mg, 0.9 mmol) in anhydrous pyridine (12ml) was added succinic anhydride (300 mg, 3 mmol). The reaction wasstirred at room temperature for 17 hours. The consumption of startingmaterial was followed by TLC using methanol-dichloromethane (1:49) asthe mobile phase. The mixture was diluted in ethyl acetate (100 ml),washed with 0.5M sodium chloride (3×100 ml) and saturated sodiumchloride (1×100 ml), and dried over anhydrous sodium sulfate. Afterconcentrating by rotary evaporation and drying under high vacuum (45°C.), 1.74 g of a yellow solid was obtained.

The yellow solid was dissolved in dry dioxane (10 ml) containinganhydrous pyridine (0.5 ml) and p-nitrophenol (350 mg, 2.5 mmol).Dicyclohexylcarbodiimide (1.0 g, 4.8 mmol) was added and the mixture wasstirred at ambient temperature. After a few minutes, dicyclohexylureabegan to precipitate. The reaction was monitored by TLC(methanol-dichloromethane, 1:9) and after 3 hours, the dicyclohexylureawas collected by filtration. Long chain alkylamine CPG (5.0 g) wassuspended in the filtrate containing the p-nitrophenyl ester derivative,triethylamine (1.0 ml) was added, and the mixture was shaken overnightat room temperature. The derivatized support was copiously washed withdimethylformamide, methanol, and diethyl ester and dried in vacuo.Before capping the unreacted alkylamine groups, the loading capacity ofthe MF-CPG® was assayed by determining the amount of dimethoxytritylcation released upon treatment with perchloric acid according topublished procedures (Oligonucleotide Synthesis: A Practical Approach,M. J. Gait (ed.), IRL Press, Oxford, 1984).

Finally, capping of MF-CPG® was achieved by treatment with aceticanhydride-pyridine-DMAP (10:90:1. v/v/w) for one hour. The support wasthoroughly washed with methanol and diethyl ether and dried under highvacuum to give 4.95 g of MF-CPG®. The capped MF-CPG® gave a negativeninhydrin test (Smith et al., supra).

3' Amine-ON™CPG, prepared by the above process, has the followingstructural formula: ##STR4##

Example 2--Synthesis of 3' Biotinylated Oligonucleotide Probes

Two 3' amino-modified oligonucleotides, GGCGCCGGCGGTGTGGGCAA-X (H-ras,wild type) and GGCGCCGGCGATGTGGGCAA-X (H-ras, codon 13 Asp) [X=3'primary amine modification], were synthesized using MP-CPG on aBiosearch Cyclone DNA synthesizer. Standard columns were packed with 1μmol of MF-CPG® and DNA synthesis was performed by suggestedmanufacturer protocols without any program changes. The couplingefficiency of the first nucleotide was determined by measuring thedeprotected dimethoxytrityl cation concentration. Solid support cleavageand deprotection were accomplished with concentrated ammonium hydroxide.

Each crude amino-modified oligonucleotide was dissolved in 0.8 ml of0.1M NaHCO₃ /NaCO₃ (pH 9). Biotin-XX-NHS ester in dimethylformamide (100mg/ml, 0.25 ml) was added and the mixture as allowed to react for 16hours at room temperature. The biotinylated probes were purified onSephadex G-25 columns (1×40 cm) and then by preparative HPLC. AnalyticalHPLC chromatograms of the purified 3' biotinylated probes are shown inFIG. 2. The presence of biotin was confirmed by ap-methylaminocinnamaldehyde colorimetric test.

Example 3--³² P 5' End Labeling of the Biotinylated OligonucleotideProbes

The H-ras biotinylated probes were ³² P 5' end labeled using amodification of the method described by Berent et al. (Berent, S. L., M.Mahmoudi, R. M. Torczynski, P. W. Bragg, and A. P. Bollon [1985]BioTechniques 3: 208-220). 100 ng of the oligonucleotide probe weredissolved in 30 μl of distilled water and heated to 65° C. for 3minutes. The oligomers were then taken up to 50 μl of a reaction bufferwhich contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 μM DTT, 0.1 mMspermidine, 10 μl gamma ³² P-ATP (specific activity 6000 Ci/mM), and15-20 units of T4 kinase. This reaction mixture was incubated at 37° C.for 30 minutes followed by the addition of another 15-30 units of kinaseand further incubation for 30 minutes. The labeled probe was thenisolated using a G- 25 Sephadex column.

Example 4--PCR Amplification of the H-ras Protooncogenes

Amplification of genomic DNA by the polymerase chain reaction (PCR) hasbeen previously described (Liu et al., supra; Saiki et al., supra). Twodifferent sets of amplifying primers (amplimers) were used to amplifyspecific ras oncogene segments of genomic DNA. ATGACGGAATATAAGCTGGT (5'H-ras amplimer) and CTCTATAGTGGGGTCGTATT (3' H-ras amplimer) were usedto amplify the region around codons 12 and 13 of the H-ras gene;ATGACTGAGTACAAACTGGT (5' N-ras amplimer) and CTCTATGGTGGGATCATATT (3'N-ras amplimer) were used to amplify the same codon region of the N-rasgene. 1 μg of genomic DNA was amplified in a 100 μl volume containing 50mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl₂, 0.1 gelatin, 0.5 mM all fourdNTPs, and 2.5 units Taq polymerase. The first amplification cycleincluded denaturation at 95° C. for 5 minutes, annealing at 55° C. for 2minutes, and primer extension at 68° C. for 2 minutes. The remaining 35cycles were performed with 2 minute incubation periods at eachtemperature.

The generation of target DNA with an H-ras codon 13 Asp mutation wasaccomplished by the method described-in Rochlitz et al. (Rochlitz, C.F., G. K. Scott, J. M. Dodson, and C. C. Benz [1988] DNA 7: 515-519). Inthis reaction, the sequence of the 3' amplifying oligomer has been notedabove. The 5' amplimer, however, encompasses the 20 nucleotide sequenceat codon 12 and 13 of H-ras and contains a point mutation encoding aglycine to aspartate change in codon 13 (GGCGCCGGCGATGTGGGCAA). DNAgenerated through PCR amplification was used as target DNA inoligonucleotide hybridization analysis. In this manner, the amplifiedDNA incorporates the oligonucleotide with the codon 13 aspartatemutation.

Example 5--Hybridization of Probe and Post Hybridization Washes withTetramethylammonium Chloride (TMAC)

40 μl of the amplified samples were added to 80 μl of 0.4N NaOH, andheated to 95° C. for 2 minutes. The reaction mixtures were neutralizedwith 100 μl of 2M Tris-HCl (pH 7.4), and the solution slotted ontoAmersham Hybond nylon filter. The DNA was crosslinked onto the filter byUV radiation under conditions suggested by the manufacturer.

The slot blots were pre-hybridized for 2 hours at 37° C. with 10-15 mlsof 5X SSPE, 5X Denhardt's, 0.5% SDS, and 100 mM sodium pyrophosphate (pH7.5). A labeled probe was then added to this solution to a finalconcentration of 5×10⁶ cpm/ml and the filters were hybridized at 50° C.for 4 to 12 hours. Following this incubation, the filters were washedonce at room temperature with 6X SSC for 20 minutes, and twice at 61° C.in 3M TMAC, 50 mM Tris-HCl (pH 8), 2 mM EDTA, and 0.1% SDS. The filterswere then washed once at room temperature with 6X SSC. Hybridization wasdetected both by autoradiography, and by colorimetric detection with astreptavidin-alkaline phosphatase conjugate (Clontech's Gene-tectSystem) on the same slot blot.

Example 6--Preparation of Non-Isotopic Oligonucleotide Labeling Kits(NIO-Label™Kits)

3' non-isotopic oligonucleotide (NIO) labeling kits are complete kitsdesigned to conveniently label synthetic oligonucleotides at the 3'terminus with either biotin or fluorescein. The labeling process isdivided into two procedures which are outlined in Sequence 1. First, anoligonucleotide is functionalized with a primary aliphatic amine at its3' terminus using 3' Amine-ON CPG (Sequence 2). 3' Amine-ON CPG is aunique CPG that incorporates a primary amine on the 3' terminus of anoligonucleotide. It is fully compatible with automated DNA synthesizers.Secondly, the 3' amino-modified oligonucleotide is reacted withBiotin-XX-NHS ester or FITC. All buffers and reagents are supplied forthis labeling procedure. Biotin-XX-NHS ester is a unique product, havingan extra long linking arm (XX) consisting of 14 atoms (Sequence 3).

List of Components

1. 3' Amine-ON CPG, four 1 μmol columns (Clontech Catalog No. 5220-1;Clontech Laboratories, Inc., Palo Alto, Calif.).

2. Biotin-XX-NHS ester, 100 mg (Clontech Catalog No. K1072-1) -or- FTIC,100 mg (Clontech Catalog No. K1073-1).

3. 10X labeling buffer, 1.0M sodium bicarbonate/carbonate (pH 9) 0.5 ml.

4. N,N-Dimethylformamide, 1.0 ml. ##STR5##

Method

Modifying the 3' Terminus with a Primary Aliphatic Amine

1. Attach a 3' Amine-ON to your automated DNA synthesizer.

2. Enter the oligonucleotide sequence you wish to synthesize. Make sureyou enter the 3' terminal base of your sequence as the 2nd base from the3' end. Note that 3' Amine-ON CPG has a bifunctional linking armattached to it instead of the 3' terminal base. Hence, the 3' base isnot on the CPG as with normal oligonucleotide synthesis. This must beaccounted for when you enter the sequence. Since automated synthesizersassume that the 3' base is attached to the CPG, a nonsense base must beentered at the 3' terminus when using 3' Amine-ON CPG.

3. Initiate the synthesis using the Trityl-Off mode, i.e., remove thefinal DMT protecting group. If desired, the primary amine incorporationcan be determined by measuring the deprotected dimethoxytritylconcentration of the first coupling cycle, typically >95%.

Deprotection

4. Cleave the 3' amino-modified oligonucleotide from the CPG by treatingit with 1 ml of ammonium hydroxide at room temperature for 2 hours. Itis convenient to use luer tip syringes for this step. Be careful not tolet the ammonia evaporate.

5. Complete the deprotection by transferring the ammonium hydroxidesolution to a 1.5 ml screw cap microcentrifuge tube and heat at 55° C.for 6 hours to overnight. Evaporate to dryness by vacuum centrifugationor rotary evaporation (Caution: ammonia gas builds up at 55° C.; cool to4° C. before opening the screw cap microcentrifuge tube). The 3'amino-modified oligonucleotide is now ready for labeling. As an option,you may wish to quantitate the primary amine groups by a publishedprocedure (Smith et al. [1985] supra).

Labeling (Agrawal, S. et al. [1986] Nucl. Acids Res. 14: 6227-6245)

6. The following reaction is designed for a 1 μmol synthesis. In a 1.5ml microcentrifuge tube, dissolve the total crude 3' amino-modifiedoligonucleotide (from step 5) in 900 μl deionized water.

7. Add 100μ of 10X labeling buffer.

8. Biotin labeling (Clontech Cat. No. K1072-1): Add 250 μl of a freshlyprepared Biotin-XX-NHS ester in N,N-dimethylformamide (DMF). Prepareimmediately before use by dissolving 25 mg of Biotin-XX-NHS ester in 250μl DMF. Vortex immediately after addition.

FITC labeling (Clontech Cat. No. K1073-1): Same procedure as with biotinlabeling except replace 25 mg Biotin-XX-NHS ester with 25 mg of FITC.

9. Incubate at room temperature overnight. When using FITC, perform thereaction in the dark. If precipitation occurs, incubate at 42° C. for 2hours.

10. Pass the reaction mixture down a 1.0 cm×40 cm column of Sephadex(Pharmacia) G25 or G50. The Sephadex column should be saturated first bypassing 1 mg of sheared, denatured salmon sperm DNA through and washingwith 100 ml of deionized water. Load sample from step 10 and elute withdeionized water. The labeled oligonucleotide will elute in the voidvolume. Collect 1 ml fractions; monitor separation via UV absorption at260 nm.

Purification

In many applications, further purification may not be necessary.However, to achieve optimum results, it is recommended to purify byRP-HPLC or electrophoresis.

Reverse phase HPLC. The lipophilic character of both biotin andfluorescein aid in oligonucleotide purification by RP-HPLC. The biotinor fluorescein labeled oligonucleotide is retained longer on the columnrequiring a higher acetonitrile concentration to elute. Thus, as thelast peak to elute, it is easily separated.

Use a RP-C18 analytical column (4.6 mm×250 mm). Set detector wavelengthto 260 nm. Use the following gradient system as a reference run: A=0.1Mtriethylammonium acetate (pH 7), B=50% acetonitrile in 0.1Mtriethylammonium acetate (pH 7); 20-80% B, 60 minutes; 1.0 ml/minute.Since retention time will vary with the size of the oligonucleotide, youmay wish to adjust the gradient after the first run.

Polyacrylamide gel electrophoresis (PAGE). Use conventional PAGE methodsfor separation of oligonucleotides, employing a 1.5 mm thick denaturing(urea) polyacrylamide gel. The percentage of acrylamide andbis-acrylamide will depend on the size of the oligonucleotidesynthesized. Load 1-2 OD labeled oligonucleotide per well.

After electrophoresis, the labeled oligonucleotide may be visualized asfollows: Remove the glass plates from the gel and wrap it in transparentplastic wrap. In a darkroom, place the gel on a fluorescent TLC plateand use a UV lamp to observe fluorescent quenching. The labeledoligonucleotide runs the slowest, since it has the highest molecularweight, and is therefore easily identified (Warning: excessive exposureto UV light may cause crosslinking of the DNA). Excise the labeledoligonucleotide with a razor blade. Crush the gel in a test tube andincubate in 0.1M ammonium bicarbonate at room temperature overnight.Finally, desalt the oligonucleotide by passing it through a Sephadex G25column.

Confirmation of Label

Biotin (Clontech Cat. No. K1070-1): The presence of biotin can bedetermined by a p-dimethylaminocinnamaldehyde colorimetric test(McCormick, C. B. and J. A. Roth [1970] Methods Enzymol. 18: 383-385).Spot 5 μl of a 30-40 OD/ml solution of the biotinylated oligonucleotideon a silica gel TLC plate. Spray with a solution of 0.2%p-dimethylaminocinnamaldehyde, 2% sulfuric acid in ethanol. Develop byslight heating. The presence of biotin is visualized by a pink-red spot.

Fluorescein (Clontech Cat. No. K1071-1): The presence of fluorescein iseasily determined by its absorption maximum at 495 nm (Smith, L. M. etal. 1985], supra). Dilute an aliquot of the FITC-labeled oligonucleotideto approximately 1.0 OD/ml (pH 7.5). Assay for the presence offluorescein by measuring absorbance in the range of 400-600 nm; anabsorbance maximum should be observed between 490 and 500 nm.

I claim:
 1. In an improved automated nucleotide synthesis processyielding an unbound oligonucleotide having a protected primary amine,sulfhydryl, disulfide, or hydroxyl attached to the 3' terminus, whereinthe improvement comprises:using a multifunctional solid support reagenthaving the structure ##STR6## wherein: C=carbon atom; W=a solid supportwhich is stable under all conditions of solid phase oligonucleotidesynthesis selected from the group consisting ofalkylamine CPG, whereinalkyl is from 1 to 50 carbon atoms, inclusive, and isomeric formsthereof; chemically modified CPG, with modifications selected from thegroup consisting of hydroxyl, carboxyl, sulfhydryl, and disulfide; andcopolymers of styrene and divinylbenzene; X=a cleavable linking armconnecting carbon C to W characterized as a combination of atom groupsthat covalently connects to the solid phase through a cleavable linkage,is stable to all the conditions of solid phase oligonucleotidesynthesis, and which is readily cleaved from the solid phase, selectedfrom the group consisting of --(CH₂)_(n) --, where n is an integer from1 to 50, --OCO--, --CO₂, --NHCO--, --CONH--, and --O--CO--CH₂ --CH₂--CO--; Y=a linking arm connecting carbon C to R₁ O-- that is at leastone carbon atom long characterized as a combination of atom groups thatcovalently connects to OR₁ and is stable to all the conditions of solidphase oligonucleotide synthesis selected from the group consisting of--(CH₂)_(n) --, where n is an integer from 1 to 50, --OCO--, --CO₂,--NHCO--, --CONH--, and --O--CO--CH₂ --CH₂ --CO--; Z=a molecule selectedfrom the group consisting of --(CH₂)_(n) --, where n is an integer from1 to 50, --OCO--, --CO₂, --NHCO--, and --CONH--, wherein said moleculeis bonded between the variable A-- and carbon C, wherein the ester andamide bonds are directed toward the A or C substituent; R₁ O=a protectedhydroxide group wherein R₁ is a base-stable/acid-labile hydroxylprotecting group selected from the group consisting of monomethoxytrityland dimethoxytrityl; A=a functional group that is capable of attaching areporter molecule or a detectable complex wherein A is selected from thegroup consisting of primary amine, sulfhydryl, disulfide, and hydroxyl;R₂ =corresponding protecting group for A that is stable to all theconditions of solid phase oligonucleotide synthesis wherein saidprotecting group is 9-fluorenylmethyl (Fmoc) or trifluoroacetyl (TFA);and X'=a molecule that is inert to solid phase oligonucleotide synthesisselected from the group consisting of H, alkyl from 1 to 50 carbonatoms, inclusive, and isomeric forms thereof, --Z--A--R₂, and --Y--OR₁.2. The process, according to claim 1, wherein said multifunctionalreagent has the following structure: ##STR7## wherein the alkyl ofalkylamine is 1 to 50 carbon atoms, inclusive, and isomeric formsthereof.
 3. The process, according to claim 1, wherein said processfurther comprises attaching a reporter molecule to said functionalgroup.
 4. The process, according to claim 1, wherein the modified 3'terminus of the synthesized oligonucleotide is cleaved from saidsupport.
 5. The process, according to claim 4, wherein said cleaving ofthe oligonucleotide from the solid support comprises the use of ammoniumhydroxide.
 6. The process, according to claim 1, wherein W is CPG. 7.The process, according to claim 1, wherein R₁ is dimethoxytrityl.
 8. Theprocess, according to claim 1, wherein R₂ is 9-fluorenylmethyl (Fmoc).9. The process, according to claim 1, wherein A is --NH--.